Peptides are heteropolymers of amino acids that are linked via their carboxyl and amino groups by amide bonds. Since the above definition holds true for proteins as well, they are often differentiated from proteins based upon their chain length, being usually described as those heteropolymers that are ranging in chain length from two to a few to several dozen amino acid residues.
Some examples of these peptides that vary vastly in their molecular weight and their functions are described here [Geysen, H. M. et al, J. Immunol. Methods, 102:259-274. (1987); Milich, D. R. Semin. Immunol., 2(5):307-315 (1990); Cochran, A. G. Chemistry & Biology, 7:R85-R94 (2000)]. The active peptide, insulin (51 residues, 5773 Da), formed after processing of the larger but inactive peptide, pro-insulin (86 residues), plays a critical role in glucose metabolism in the body and lack of it leads to type I diabetes. Parathyroid hormone (84 residues) is the most important endocrine regulator of the levels of calcium and phosphorus in the blood. A much smaller version of the parathyroid hormone, teriparatide (34 residues), is sold by Eli Lilly for the treatment of osteoporosis. Calcitonin (32 residues) also plays a critical role in calcium metabolism and has opposite effects with respect to the parathyroid hormone. It is also used in the treatment for osteoporosis. Angiotensin I, II, III and IV having 10, 8, 7 and 6 residues respectively, have diverse roles as endocrine, paracrine, and intracrine hormones, causing vasoconstriction, increasing blood pressure and causing release of aldosterone from the adrenal cortex. The non-caloric sweetener, aspartame (a dipeptide of aspartic acid and esterified phenylalanine) is used as a sugar substitute. These examples illustrate the vast diversity of functions, chain length and utility amongst peptides. Their role as mediators of key biological functions e.g., as hormones, enzyme substrates or inhibitors, neurotransmitters, immunomodulators and, antiviral agents make them particularly attractive as therapeutic agents. More than 40 therapeutic peptides are available in the world market today and more than 400 peptides are in advanced pre-clinical phases of drug development worldwide [Parmar, H. Therapeutic Peptides in Europe: Finding the Opportunities, Frost and Sullivan Market Insight, Nov. 26, 2004]. Therefore the therapeutic application of peptides has an enormous potential.
Originally peptides of therapeutic significance used to be isolated from biological tissues. For example, insulin was produced from the ox pancreas [Collip, J. J. Physiol., 66:416-430 (1928)], and Calcitonin from the ultimobronchial glands of fish [(Parkes, Colo. et al., Fed. Proc., 28:413 (1969)]. The tissue origin of these peptides made the isolation methods difficult and cumbersome, yielded non-ideal product purity, carried the risk of transmitting infections and generally affected the commercial scalability, ultimately limiting the commercialization of peptides as therapeutics.
Chemical method is one of the solutions to the above problem of commercial production—at least for the production of small and medium sized peptides ranging from about 5 to 80 residues [Kimmerlin, T. and Seebach, D. J. Pept. Res., 65(2):229-260 (2005)]. But this method also has many disadvantages that make it inefficient in cost, such as, the possibility of racemization, poor solubility of protected peptide fragments, limitation on the length of peptide and serial side reactions. The undesirable side reactions associated with this method decrease the yield and render necessary, difficult and lengthy purification procedures. Therefore, even though chemical synthesis is the most mature technology for peptide synthesis today, it is fraught with problems that add to the overall cost of their production signifying the need for developing other distinctly different methods of their production.
In principle a large number of the above problems can be solved by employing the recombinant DNA technology that allows the selection, amplification and manipulation of expression of endogenous and foreign genes in microbial cells. This technology is better than the chemical technology in that it naturally produces non-racemic and fully correct a.a. sequences, as nature has evolved living organisms to do so. Further this technology also allows more environmentally friendly processes of production and purification. However, this technology is limited by the fact that in general, it is not possible to get high levels of expression of peptides in a microbial host. Because of their short length, peptides get easily degraded by the E. coli proteolytic machinery [Marston, F. A. Biochem. J., 240:1-12 (1986); Makrides, S. C. Microbiol. Rev., 60:512-538 (1996)]. Many eukaryotic foreign peptides are recognized as abnormal in Escherichia coli and get rapidly degraded in the expression host [Goldschmidt, R. Nature, 228:1151-1154 (1970); Lin, S. & Zabin, I. J. Biol. Chem., 247:2205-2211 (1972)]. The half-life of human proinsulin in E. coli has been reported to be only 2 minutes [Talmadge, K. & Gilbert, W. Proc. Natl. Acad. Sci. USA, 79:1830-1833 (1982)].
A common strategy to overcome the problem of degradation of peptides in the expression host is to express them as fusion proteins in conjunction with another larger peptide or protein, which acts as a fusion partner. After expression, the peptide and the fusion partner are separated from each other by chemical or proteolytic cleavage at a site which was pre-designed into the fusion product. In this regard many proteins have been used as fusion partners that produce the fusion product in both soluble and insoluble form inside the cell. Ray et al., [Ray, M. V. L., et al., Bio/Technology, 11:64-70 (1993)] describe the use of glutathione-S-transferase (GST) as a fusion partner to get soluble intracellular expression in E. coli of salmon calcitonin which was cleaved away from the fusion protein with cyanogen bromide. Dykes et al., [Dykes, C. W. et al., Eur. J. Biochem., 174:411-416 (1988)] describes the soluble intracellular expression in E. coli of a fusion protein consisting of a human atrial natriuretic peptide and chloramphenicol acetyltransferase, where the fusion protein was either proteolytically cleaved by thrombin or enterokinase or chemically cleaved with 2-(2-nitrophenylsulphenyl)-methyl-3′-bromoindolenine to release the peptide of interest. Maltose-binding protein and thioredoxin have also been used as solubilizing fusion partners [Baneyx, F. Curr. Opin. Biotechnol., 10:411-421 (1999)]. Another example is that of U.S. Pat. No. 5,223,404 which describes the use of ompA as a fusion partner with PTH (1-34) in order to get the expression of soluble protein in periplasmic space.
While in many cases the soluble fusion product yields good expression [Cipakova, I. et al., Protein Expr. Purif, 37:207-212 (2004); Forrer, P. & Jaussi, R. Gene, 224:45-52 (1998); Hoffman, F. et al., Enz. and Microb. Technol., 34:235-241 (2004); Baneyx, F. Curr. Opin. Biotechnol., 10:411-421 (1999)] and also keeps the fused protein in its correct folding conformation [Marco, V. D. et al., Biochem. Biophys. Res. Commun. 322:766-771 (2004); Zou, Z. et al., Journal of Biotechnology, 135:333-339, (2008)] this technology has remained commercially unattractive. This is because in the cytoplasm the product is present along with hundreds or thousands of other host proteins making the process of purification extremely expensive and inefficient. On the other hand soluble expression in the periplasm is limited by the small size of this space. Therefore, it is not surprising to note that commercial examples of soluble expression of peptides are rare, if not entirely non-existent. To circumvent these problems peptides are often produced as insoluble fusion products that form inclusion bodies due to the precipitation and aggregation of the proteins with in the cell [Williams, D. C. et al., Science 215:687-688 (1982)]. The formation of inclusion bodies provides several advantages for commercial production of proteins. The inclusion bodies not only prevent the degradation of the expressed protein by protecting the product of interest from the action of cellular proteases (Singh, S. M. and Panda, A. K., J. Biosci. Bioengg. 99(4):303-310 (2005)], but also ensure that the product is produced in relatively very high level of purity [Panda, A. K. Adv. Biochem. Engg./Biotechnol. 85:43-93 (2003)]. The difference in size and density of inclusion bodies as compared to cells results in easy isolation of these inclusion bodies from the cells [Bowden, G. A. et al., Bio/Technol. 9:725-730 (1991)]. Also the homogeneity of the protein of interest in inclusion bodies helps in decreasing the number of purification steps to recover the pure protein [Panda, A. K. Adv. Biochem. Engg./Biotechnol. 85:43-93 (2003)]. The inclusion body protein also minimizes the toxic effects of the expressed recombinant protein to E. coli cells [Lee, J. et al., Biochem. Biophys. Res. Commun., 277:575-580 (2000); Lee, J. et al, Appl. Microbiol. Biotechnol., 58:790-796 (2002); Wei, Q. et al., Appl. Environ. Microbiol., 71(9):5038-5043 (2005); Rao, X. C. et al., Protein Expr. Purif, 36:11-18 (2004)]. Overall, the expression of target protein in the form of inclusion bodies ensures that the target protein is produced economically [); Fahnert, B. et al., Adv. Biochem Engg./Biotechnol. 89:93-142 (2004); Walsh, G. Nat. Biotechnol. 21:865-870 (2003); Mukhopadhyay, A. Adv. Biochem. Engg./Biotechnol. 56:61-109 (1997)].
In fact many major biotech companies have been using fusion proteins to express therapeutic peptides in the form of inclusion bodies. In EP 0055945, Genentech reports the use of TrpE and B-galactosidase (lacZ) as a fusion partner to express proinsulin, which is later cleaved off using cyanogen bromide. In EP0211299 A, Hoechst AG, describes the use of D peptide of E. coli Trp gene as a fusion partner to express fusion peptides proinsulin and hirudin. In U.S. Pat. No. 5,670,340, Suntory Ltd, Osaka, Japan, reports the use of a fragment of beta galactosidase as a fusion partner along with a leader peptide linked with human calcitonin to express the fusion product as inclusion bodies, where calcitonin is cleaved off using V8 protease after expression. In U.S. Pat. No. 6,500,647, Mogam Biotechnology Institute, exemplified the production of human PTH as inclusion bodies by fusing it to a phosphoribulokinase gene fragment of Rhodobacter sphaeroides or its mutated gene as a fusion partner where the fusion product is also containing a urokinase-specific cleavage site. [Wingender, E. et al., J. Biol. Chem. 264(8):4367-4373 (1989)] describes the use of variable sizes of cro-beta galactosidase as a fusion partner with PTH which is later cleaved off using acid hydrolysis. Despite the above described successes associated with the use of fusion partners for the production of therapeutic peptides, literature sites a number of problems that still exist with this technology. Some of the examples below illustrate these problems and support the need for inventing better fusion partners.
Not all fusion partners form stable inclusion bodies. Shen [Shen, S-H. PNAS, 81:4627-4631 (1984)], describes the bacterial expression of proinsulin using an 80 a.a. long, amino-terminus fragment of β-galactosidase as a fusion partner. This fusion partner resulted in extremely unstable expression of the fusion product not detectable in SDS-PAGE. Stable expression in the form of inclusion bodies was obtained only when two tandem copies of pro-insulin gene were used in conjunction with the above described fusion partner. Therefore an ideal size of the fusion partner appears to be an important criteria for the formation of inclusion bodies.
While often the formation of inclusion bodies is a consequence of high expression rates [Kane, J. F. and Hartley, D. L. Trends Biotechnol. 6:95-101 (1988); Fahnert, B. et al., Adv. Biochem Engg./Biotechnol. 89:93-142 (2004)], the literature teaches us that such a correlation is not always correct. Mac [Mac, T-T. Towards solid-state NMR spectroscopic studies of the ETBR/ET-1 complex. Ph.D. Thesis (2007), Freie Universitat Berlin] used thioredoxin as a fusion partner for the expression of BigET-1 peptide with the intention that the small size of thioredoxin (12 kDa) and its solubility in E. coli will ensure a soluble fusion product. In contrast to this expectation, the fusion product formed inclusion bodies but at very poor expression levels (45 mg/L). When GST was used a fusion protein for the expression of ET-1 in the form of inclusion bodies, expression levels of the fusion product increased but still remained in mg/L levels only. Use of beta-galactosidase fragments as a fusion partner also led to the expression of ET-1 in the form of inclusion bodies but the expression levels remained low, yielding only few mg/L quantities of ET-1 at the end of the process [Ohashi, H. et at., Appl. Microbiol. Biotechnol. 41:677-683 (1994); Yasufuku, K. et al., J. Biochem. 112:360-365 (1992)]. Therefore just the ability of a fusion partner to induce inclusion bodies does not ensure high expression and subsequent commercial viability.
The vast numbers of above references indicate that the production of small peptides from bacteria has been problematic for a variety of reasons. While the problem of proteolysis of peptides in microbial cells is usually taken care of quite successfully by expressing them as fusion proteins but making these processes commercially feasible has been fraught with numerous challenges. While expressing peptides as insoluble fusion proteins is a preferred method of commercial production of peptides by recombinant methods, the choice of a fusion partner is not straight forward. The previously reported fusion proteins do not always behave in a predictable fashion as far as formation of inclusion bodies and/or high and stable expression is concerned. Further, many of them are either not very easily available through commercial sources or their use for commercial production of a desired peptide has not been fully established. Hence there has been a long felt need in the art for a suitable expression system that comprises a fusion partner that would consistently give stable and high expression with a variety of peptides and at a low cost. With this view, the object of the present invention is to develop an expression system comprising such a fusion partner and establish a production method for the production of peptides at a high yield and production efficiency which is equal to or better than those of the existing processes.
The present invention discloses a novel expression system that utilizes a novel fusion partner, G-CSF, which can be consistently used for the high expression of peptides in the form of inclusion bodies. The peptides of interest that can be expressed with this fusion partner, vary not only in their amino acid content but also in their chain length, and are separated from the fusion partner after cleaving the cleavage site, which is pre-designed into the fusion product. The fusion peptide obtained as such, may then be purified using the standard downstream purification methods. The process of production utilizing such an expression system was found to be highly scalable and facilitated in the stable high expression for three peptides that it was tested with.